title: Tailing DNA aptamers with a functional protein by two-step enzymatic reaction.
abstract: An efficient, quantitative synthetic strategy for aptamer-enzyme conjugates was developed by using a two-step enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT) was used to first incorporate a Z-Gln-Gly (QG) modified nucleotide which can act as a glutamine donor for a subsequent enzymatic reaction, to the 3'-OH of a DNA aptamer. Microbial transglutaminase (MTG) then catalyzed the cross-linking between the Z-QG modified aptamers and an enzyme tagged with an MTG-reactive lysine containing peptide. The use of a Z-QG modified dideoxynucleotide (Z-QG-ddUTP) or a deoxyuridine triphosphate (Z-QG-dUTP) in the TdT reaction enables the controlled introduction of a single or multiple MTG reactive residues. This leads to the preparation of enzyme-aptamer and (enzyme)n-aptamer conjugates with different detection limits of thrombin, a model analyte, in a sandwich enzyme-linked aptamer assay (ELAA). Since the combination of two enzymatic reactions yields high site-specificity and requires only short peptide substrates, the methodology should be useful for the labeling of DNA/RNA aptamers with proteins.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Takahara
authors.forename: Kounosuke
authors.initials: K
authors.lastname: Hayashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D003856:Deoxyuracil Nucleotides; D004151:Dipeptides; D011994:Recombinant Proteins; C432806:glutaminyl-glycine; C027078:deoxyuridine triphosphate; D011503:Transglutaminases; D013917:Thrombin
keywords: DNA aptamer; DNA-enzyme conjugate; Enzyme-linked aptamer assay; Microbial transglutaminase; Terminal deoxynucleotidyl transferase; Thrombin